Our long-term goal is to identify effective therapeutic targets for preventing neurodegeneration in Alzheimer's disease (AD). In this proposal, we investigate a novel mechanism of AD pathogenesis following our discovery of transcription factors FOXO1 and FOXO3a as novel Cyclin Dependent Kinase-5 (Cdk5) substrates. FOXO1 and FOXO3a are highly expressed in the brain, specifically in areas susceptible to neurodegeneration in AD. However, the roles and regulation of FOXOs in AD are unclear. The activation of FOXOs and their post- translational modifications have not been analyzed in human clinical samples. We show that the activation of FOXOs occurs early in AD clinical tissues and is highly neurotoxic in b-amyloid-mediated signaling. Further, our data suggest that FOXOs are activated by two independent mechanisms: (i) Cdk5-mediated phosphorylation (FOXO1 at S249, FOXO3a at S173); (ii) calcineurin-mediated dephosphorylation of FOXOs at Akt sites. Akt is known to phosphorylate FOXO1 and FOXO3a, rendering them inactive. Hypothesis: Cdk5 and calcineurin synergistically promote FOXOs' activation, which leads to neurotoxic b- amyloid processing and cell death, two hallmarks of AD. The impact of this work is that its successful completion will provide retrospective biomarkers and potential new strategies for AD treatment. This hypothesis will be tested by pursuing three specific aims: Aim 1: Dissect the molecular mechanism by which FOXOs are activated by Cdk5 and calcineurin in rat and human fetal neurons. Aim 2: Determine the roles of active FOXOs in promoting b-amyloid processing and neurotoxicity in rat and human fetal neurons. Aim 3: Investigate the clinical relevance of FOXOs' phosphorylation in two AD mouse models (APP/PS1 and p25-transgenic mice) and human clinical tissues. While APP/PS1 mice will confirm a global role of Ab in FOXO signaling, p25-mice will specifically demonstrate Cdk5's role in activating FOXOs and their correlation with neurodegeneration. Innovation: The hypothesis is formulated based on novel Cdk5 substrates FOXO1 and FOXO3a, discovered using a highly innovative chemical genetic approach. Second, this study suggests that inhibiting FOXOs directly or targeting the upstream regulators of FOXO signaling will provide novel therapeutic intervention points for preventing neurodegeneration in AD. Third, our study provides a novel molecular link between insulin depletion (a cause for AD), Akt inhibition, activation of FOXOs and neurodegeneration in Ab1-42 neurotoxicity. Significance: We propose that activating Akt or inhibiting calcineurin and Cdk5 will abrogate FOXO signaling, causing neuroprotection in AD. Analysis of FOXO post-translational modifications coupled with Cdk5, CaN and Akt activation levels in AD mouse models and human clinical samples will aid in the development of novel tools for retrospective analysis to better understand AD pathogenesis. Thus, we expect that determining the molecular mechanisms by which FOXO transcriptional pathway contributes to disease pathogenesis will be an important step forward in AD prevention and treatment.